Impact of video games on plasticity of the hippocampus.

The hippocampus is critical to healthy cognition, yet results in the current study show that action video game players have reduced grey matter within the hippocampus. A subsequent randomised longitudinal training experiment demonstrated that first-person shooting games reduce grey matter within the hippocampus in participants using non-spatial memory strategies. Conversely, participants who use hippocampus-dependent spatial strategies showed increased grey matter in the hippocampus after training. A control group that trained on 3D-platform games displayed growth in either the hippocampus or the functionally connected entorhinal cortex. A third study replicated the effect of action video game training on grey matter in the hippocampus. These results show that video games can be beneficial or detrimental to the hippocampal system depending on the navigation strategy that a person employs and the genre of the game.

Vacuolar myelopathy in transgenic mice expressing human immunodeficiency virus type 1 proteins under the regulation of the myelin basic protein gene promoter.

Vacuolar myelopathy is a common neurological complication in AIDS patients. The pathogenesis of this spinal cord white matter disease remains unclear and it is still debated whether infection of spinal cord with the human immunodeficiency virus type 1 (HIV-1) is causing the disease. We have generated transgenic mice expressing the entire HIV-1 genome under the regulation of an oligodendrocyte-specific promoter. These mice develop spinal cord vacuolar lesions similar to those found in AIDS patients. This animal model provides in vivo evidence linking the expression of HIV-1 proteins in oligodendrocytes to the spinal cord damage found in vacuolar myelopathy.

Prevention of breast tumour development in vivo by downregulation of the p185neu receptor.

Certain strains of transgenic mice that express the rat neu oncogene (neuT) in mammary epithelial cells develop breast tumours at an average of 44 weeks of age. In this study, intraperitoneal injection of a monoclonal anti-receptor antibody specific for the rat neuT oncogene product dramatically affected tumour development in these transgenic mice in a dose-dependent manner. A significant proportion (50%) of mice, when injected with anti-receptor antibodies, did not develop tumours even after 90 weeks of age. The phosphotyrosine levels of the membrane fraction of breast tissues in the anti-receptor antibody-treated mice were almost completely abolished when a higher dose of antibodies was used. This study demonstrates, for the first time, that immunologic manipulation of an oncogene product can effectively prevent the development of tumours in a rodent transgenic model.

Transcription factor SCL is required for c-kit expression and c-Kit function in hemopoietic cells.

In normal hemopoietic cells that are dependent on specific growth factors for cell survival, the expression of the basic helix-loop-helix transcription factor SCL/Tal1 correlates with that of c-Kit, the receptor for Steel factor (SF) or stem cell factor. To address the possibility that SCL may function upstream of c-kit, we sought to modulate endogenous SCL function in the CD34(+) hemopoietic cell line TF-1, which requires SF, granulocyte/macrophage colony-stimulating factor, or interleukin 3 for survival. Ectopic expression of an antisense SCL cDNA (as-SCL) or a dominant negative SCL (dn-SCL) in these cells impaired SCL DNA binding activity, and prevented the suppression of apoptosis by SF only, indicating that SCL is required for c-Kit-dependent cell survival. Consistent with the lack of response to SF, the level of c-kit mRNA and c-Kit protein was significantly and specifically reduced in as-SCL- or dn-SCL- expressing cells. c-kit mRNA, c-kit promoter activity, and the response to SF were rescued by SCL overexpression in the antisense or dn-SCL transfectants. Furthermore, ectopic c-kit expression in as-SCL transfectants is sufficient to restore cell survival in response to SF. Finally, enforced SCL in the pro-B cell line Ba/F3, which is both SCL and c-kit negative is sufficient to induce c-Kit and SF responsiveness. Together, these results indicate that c-kit, a gene that is essential for the survival of primitive hemopoietic cells, is a downstream target of the transcription factor SCL.

The effect of anti-neoplastic drugs on murine acquired immunodeficiency syndrome.

The murine acquired immunodeficiency syndrome (MAIDS) is associated with proliferation of target cells that have been infected by a defective retrovirus. To control the growth of this primary neoplasia, virus-inoculated mice were treated with anti-neoplastic drugs. Paradoxically, cyclophosphamide, which is also immunosuppressive, was very effective in preventing the appearance and progression of the disease, in restoring a normal T cell function, and in depleting the number of infected target cells. This result suggests that the proliferating infected target cells were responsible for the immunodeficiency.

Immunodeficiency and clonal growth of target cells induced by helper-free defective retrovirus.

The murine acquired immunodeficiency syndrome is induced by a defective retrovirus. To study the role of virus replication in this disease, helper-free stocks of defective Duplan virus were produced. These stocks were highly pathogenic in absence of detectable replicating murine leukemia viruses (MuLVs) other than xenotropic MuLV. They induced expansion of the infected cell population (over 1000-fold), and this cell expansion was oligoclonal in origin and, most likely, arose through cell division. These results suggest that this defective virus is oncogenic, inducing a primary neoplasia associated with an acquired immunodeficiency syndrome as a paraneoplastic syndrome. These data emphasize the need to determine whether virus replication is necessary for the progression of other immunodeficiency diseases, including acquired immunodeficiency syndrome, and whether these diseases also represent paraneoplastic syndromes.

Dysregulated expression of mitotic regulators is associated with B-cell lymphomagenesis in HOX11-transgenic mice.

Dysregulated expression of the homeobox gene, HOX11 is a frequent etiologic event in T-cell acute lymphoblastic leukemias. HOX11-transgenic mice (IgHmu-HOX11Tg)-expressing HOX11 in the B-cell compartment develop B-cell lymphomas with extended latency. The latency suggests that additional genetic events are required prior to the onset of malignant lymphoma. We report the identification of 17 HOX11 collaborating genes, revealed through their propensity to be targeted in a proviral insertional mutagenesis screen. Seven integrations disrupted genes in mitotic spindle checkpoint control, suggesting that cells with elevated HOX11 expression are especially sensitive to dysregulation of chromosome segregation during mitosis. IgHmu-HOX11Tg primary B-lymphocyte cultures exposed to the aneugenic agents, colchicine and colcemid, exhibited increased incidences of chromosome missegregation as assessed by cytokinesis-block micronucleus assays. Additionally, IgHmu-HOX11Tg cultures were shown to exhibit aberrant bypass of spindle checkpoint arrest, as assessed by the increased presence of cycling cells determined by assessment of DNA content and by BrdU immunolabelling. Western immunoblotting revealed elevated expression of the mitotic effector molecules, cyclin A, cyclin B1 and cdc20 in IgHmu-HOX11Tg cultures. Moreover, spontaneously arising lymphoid neoplasms in IgHmu-HOX11Tg mice frequently exhibit aberrant expression of mitotic regulators, concomitant with increased development of micronuclei, abnormal mitotic checkpoint control and increased incidences of abnormal karyotypes when expanded in culture. Collectively, these findings indicate that abnormal regulation of spindle checkpoint control as a result of HOX11 overexpression leads to a heightened predisposition for development of aneuploidy, contributing to oncogenesis.

Strong selection for cells containing new ecotropic recombinant murine leukemia virus provirus after propagation of C57BL/6 radiation-induced thymoma cells in vitro or in vivo.

Using the Southern procedure, we have studied the presence of ecotropic-specific murine leukemia viral sequences in genomic DNA isolated from primary X-ray-induced thymomas, from lymphoid cell lines established from them, or from secondary tumors passaged in vivo. We found that primary radiation-induced thymomas and infiltrated spleens do not harbor newly acquired ecotropic provirus. However, additional ecotropic proviruses (which appear recombinant in the gagpol region) could be detected in most of the tumorigenic cell lines established in vitro from them and in tumors arising from subcutaneous transplantation of the primary thymomas. These results suggest that primary radiation-induced thymomas may not be clonal. They also indicate a strong correlation between the presence of ecotropic recombinant proviruses in the genome and the growth ability, both in vitro and in vivo, of specific cells within these thymomas, suggesting a possible mitogenic function for murine leukemia virus.

Specific expression of the human CD4 gene in mature CD4+ CD8- and immature CD4+ CD8+ T cells and in macrophages of transgenic mice.

The CD4 protein plays a critical role in the development and function of the immune system. To gain more insight into the mechanism of expression of the human CD4 gene, we cloned 42.2 kbp of genomic sequences comprising the CD4 gene and its surrounding sequences. Studies with transgenic mice revealed that a 12.6-kbp fragment of the human CD4 gene (comprising 2.6 kbp of 5' sequences upstream of the transcription initiation site, the first two exons and introns, and part of exon 3) contains the sequences required to support the appropriate expression in murine mature CD4+ CD8- T cells and macrophages but not in immature double-positive CD4+ CD8+ T cells. Expression in CD4+ CD8+ T cells was found to require additional regulatory elements present in a T-cell enhancer fragment recently identified for the murine CD4 gene (S. Sawada and D. R. Littman, Mol. Cell. Biol. 11:5506-5515, 1991). These results suggest that expression of CD4 in mature and immature T-cell subsets may be controlled by distinct and independent regulatory elements. Alternatively, specific regulatory elements may control the expression of CD4 at different levels in mature and immature T-cell subsets. Our data also indicate that mouse macrophages contain the regulatory factors necessary to transcribe the human CD4 gene.

An X-linked gene affecting mouse cell DNA synthesis also affects production of unintegrated linear and supercoiled DNA of murine leukemia virus.

To identify specific cellular factors which could be required during the synthesis of retroviral DNA, we have studied the replication of murine leukemia virus in mouse cells temperature sensitive for cell DNA synthesis (M. L. Slater and H. L. Ozer, Cell 7:289-295, 1976) and in several of their revertants. This mutation has previously been mapped on the X chromosome. We found that a short incubation of mutant cells at a nonpermissive temperature (39 degrees C) during the early part of the virus cycle (between 0- to 20-h postinfection) greatly inhibited virus production. This effect was not observed in revertant or wild-type cells. Molecular studies by the Southern transfer procedure of the unintegrated viral DNA synthesized in these cells at a permissive (33 degrees C) or nonpermissive temperature revealed that the levels of linear double-stranded viral DNA (8.8 kilobase pairs) were nearly identical in mutant or revertant cells incubated at 33 or 39 degrees C. However, the levels of two species of supercoiled viral DNA (with one or two long terminal repeats) were significantly lower in mutant cells incubated at 39 degrees C than in mutant cells incubated at 33 degrees C or in revertant cells incubated at 39 degrees C. Pulse-chase experiments showed that linear viral DNA made at 39 degrees C could not be converted into supercoiled viral DNA in mutant cells after a shift down to 33 degrees C. In contrast, such conversion was observed in revertant cells. Restriction endonuclease analysis did not detect differences in the structure of linear viral DNA made at 39 degrees C in mutant cells as compared to linear viral DNA isolated from the same cells at 33 degrees C. However, linear viral DNA made at 39 degrees C in mutant cells was poorly infectious in transfection assays. Taken together, these results strongly suggest that this X-linked gene, affecting mouse cell DNA synthesis, is operating in the early phase of murine leukemia virus replication. It seems to affect the level of production of unintegrated linear viral DNA only slightly while greatly reducing the infectivity of these molecules. In contrast, the accumulation of supercoiled viral DNA and subsequent progeny virus production are greatly reduced. Our pulse-chase experiments suggest that the apparent, but not yet identified, defect in linear viral DNA molecules might be responsible for their subsequent impaired circularization.

Two distinct Notch1 mutant alleles are involved in the induction of T-cell leukemia in c-myc transgenic mice.

We have previously characterized a large panel of provirus insertion Notch1 mutant alleles and their products arising in thymomas of MMTV(D)/myc transgenic mice. Here, we show that these Notch1 mutations represent two clearly distinct classes. In the first class (type I), proviral integrations were clustered just upstream of sequences encoding the transmembrane domain. Type I Notch1 alleles produced two types of mutant Notch1 RNA, one of which encoded the entire Notch1 cytoplasmic domain [N(IC)] and the other of which encoded a soluble ectodomain [N(EC)(Mut)] which, in contrast to the processed wild-type ectodomain [N(EC)(WT)], did not reside at the cell surface and became secreted in a temperature-dependent manner. A second, novel class of mutant Notch1 allele (type II) encoded a Notch1 receptor with the C-terminal PEST motif deleted (DeltaCT). The type II Notch1(DeltaCT) protein was expressed as a normally processed receptor [N(EC)(WT) and N(IC)(DeltaCT)] at the cell surface, and its ectodomain was found to be shed into the extracellular medium in a temperature- and calcium-dependent manner. These data suggest that both type I and type II mutations generate two structurally distinct Notch1 N(EC) and N(IC) proteins that may participate in tumor formation, in collaboration with the c-myc oncogene, through distinct mechanisms. Constitutive type I N(IC) and type II N(IC)(DeltaCT) expression may enhance Notch1 intracellular signaling, while secreted or shed type I N(EC)(Mut) and type II N(EC) proteins may differentially interact in an autocrine or paracrine fashion with ligands of Notch1 and affect their signaling.

Proviral integration site Mis-1 in rat thymomas corresponds to the pvt-1 translocation breakpoint in murine plasmacytomas.

Two loci independently implicated in T-and B-lymphocyte neoplasia are shown to be equivalent. The Mis-1 locus is a common proviral integration site in retrovirally induced rat T lymphomas, while the pvt-1 locus on murine chromosome 15 frequently translocates to the kappa locus in plasmacytomas bearing 6;15 translocations. By comparing cloned sequences, we show that pvt-1 is the murine homolog of Mis-1.

Identification of a new common provirus integration site in gross passage A murine leukemia virus-induced mouse thymoma DNA.

The Gross passage A murine leukemia virus (MuLV) induced T-cell leukemia of clonal (or oligoclonal) origin in inoculated mice. To study the role of the integrated proviruses in these tumor cells, we cloned several newly integrated proviruses (with their flanking cellular sequences) from a single tumor in procaryotic vectors. With each of the five clones obtained, a probe was prepared from the cellular sequences flanking the provirus. With one such probe (SS8), we screened several Gross passage A MuLV-induced SIM.S mouse tumor DNAs and found that, in 11 of 40 tumors, a provirus was integrated into a common region designated Gin-1. A 26-kilobase-pair sequence of Gin-1 was cloned from two lambda libraries, and a restriction map was derived. All proviruses were integrated as a cluster in the same orientation within a 5-kilobase-pair region of Gin-1, and most of them had a recombinant structure of the mink cell focus-forming virus type. The frequency of Gin-1 occupancy by provirus was much lower in thymoma induced by other strains of MuLV in other mouse strains. Using somatic-cell hybrid DNAs, we mapped Gin-1 on mouse chromosome 19. Gin-1 was not homologous to 16 known oncogenes and was distinct from the other common regions for provirus integration previously described. Therefore, Gin-1 appears to represent a new common provirus integration region. The integration of a provirus within Gin-1 might be an important event leading to T-cell transformation, and the Gin-1 region might harbor sequences which are involved in tumor development.

A viral long terminal repeat expressed in CD4+CD8+ precursors is downregulated in mature peripheral CD4-CD8+ or CD4+CD8- T cells.

The long terminal repeat from a thymotropic mouse mammary tumor virus variant, DMBA-LV, was used to drive the expression of two reporter genes, murine c-myc and human CD4, in transgenic mice. Expression was observed specifically in thymic immature cells. Expression of c-myc in these cells induced oligoclonal CD4+ CD8+ T-cell thymomas. Expression of human CD4 was restricted to thymic progenitor CD4- CD8- and CD4+ CD8+ T cells and was shut off in mature CD4+ CD8- and CD4- CD8+ T cells, known to be derived from the progenitor double-positive T cells. These results suggest the existence of similar and common factors in CD4+ CD8- and CD4- CD8+ T cells and support a model of differentiation of CD4+ CD8+ T cells through common signal(s) involved in turning off the expression of the CD4 or CD8 gene.

Transgenic mice carrying the mouse mammary tumor virus ras fusion gene: distinct effects in various tissues.

Transgenic mice carrying the v-Ha-ras oncogene under the control of the mouse mammary tumor virus long terminal repeat were produced. These mice exhibit several phenotypes: mammary tumors, bilateral hyperplasia of the harderian lacrimal gland, primary bronchio-alveolar lung adenocarcinoma, and splenomegaly. High levels of the transgene RNA were detected in mammary, harderian, and lung tumors. Accumulation of cells of the myeloid lineages was found in enlarged spleens. This phenotype may represent an indirect effect of v-Ha-ras expression on myeloid progenitors. Our data illustrate the cell-specific effects of v-Ha-ras.

Leukemogenicity of clonal isolates of murine leukemia viruses.

The leukemogenicity of three types of cloned, in vitro grown murine retroviruses was studied. Two Moloney virus clones caused leukemia, as did five clones of the B-tropic endogenous virus of BALB/c mice. Neither of two clones of N-tropic BALB/c virus caused leukemia in Fv-1n/n mice, and the viruses were not recoverable from the animals. The ability to induce leukemia therefore appeared to reside in the genome of at least certain nondefective murine retroviruses.

Elevated frequency of loss of heterozygosity in mammary tumors arising in mouse mammary tumor virus/neu transgenic mice.

Loss of heterozygosity (LOH) analysis was performed on 62 mammary tumors that were induced in (BALB/c x C57BL/6)F1 mouse mammary tumor virus/neu transgenic mice. Eighty-six simple sequence length polymorphism markers were used to cover all of the somatic chromosomes. Frequency of LOH was observed to be significant for chromosomes 4 (50%), 19 (32%), and 8 (21%). On chromosome 4, at least three distinct regions of allelic deletions could be identified: one proximal to 22 cM; the second close to the p16INK4a/p15INK4b locus, which is commonly deleted in various tumors; and the third one in the proximity of Mom1. The frequency of LOH on chromosome 19 was the same for the four markers used. Our data suggested the presence of two distinct LOH loci, one proximal to 47 cM and the other at the distal region. On chromosome 8, possibly two distinct LOH loci could be recognized, one around 52 cM and the other one at 67 cM or distal to it. These regions map close to E-cadherin (Cdh1) and M-cadherin (Cdh15) loci, respectively. Because LOH sites are thought to harbor tumor suppressor genes, this allelotype screening has allowed the mapping of putative tumor suppressor genes that may be implicated, in collaboration with the erbB-2/neu oncogene, in the development of mammary tumors in these transgenic mice.

Fos proteins can act as negative regulators of cell growth independently of the fos transforming pathway.

The proto-oncogene c-fos is known to be an important positive regulator of cell growth and notably of the G0/G1 transition. However, we observed that v-fos or c-fos-transformed rat-1 fibroblasts paradoxically had a low growth rate as compared to control untransformed rat-1 cells. We determined that this slow growth mainly reflects an increase of the G1 phase of the cell cycle (up to fourfold). In addition, the G0 --> S progression of serum-starved fos-expressing rat-1 cells refed with serum was found to be also delayed as compared to rat-1 cells. The delayed G0 --> S progression in fos-expressing cells was accompanied by the inappropriate levels or kinetics of expression of several cell cycle-regulated genes (cyclin D1, cdc2, cdk2, cdk4 and rb). Furthermore, a clear uncoupling of the pRb hyperphosphorylation with the entry into S phase was found in these fos-expressing rat-1 cells. Interestingly, the effect of the Fos proteins on the cell cycle was independent of the fos transforming pathway, indicating that the effector genes for Fos proteins are likely to be different for each process. In conclusion, our results indicate that Fos proteins may act as negative regulators of cell growth in some cell types, independently of the fos transforming pathway.

A full-length Notch1 allele is dispensable for transformation associated with a provirally activated truncated Notch1 allele in Moloney MuLV-infected MMTV(D)/myc transgenic mice.

The Notch1 gene was previously found to be targetted by provirus insertion in a high proportion of T-cell lymphomas arising in Moloney MuLV-inoculated MMTV(D)/myc transgenic mice. Proviral activation of Notch1 was associated with overexpression of truncated Notch1, deleted of the sequences coding for the extracellular domain. The high levels of truncated Notch1 RNA and proteins in these tumors are thought to be involved in the oncogenic transformation. However, in addition to these truncated RNA and proteins, high level expression of full-length Notch1 RNA and proteins was also observed in several tumors, suggesting that they could also contribute to the transformation process. To test this hypothesis, we used a genetic approach and studied MMTV(D)/myc transgenic mice in which one of the Notch1 alleles was mutated by targeted mutagenesis (Notch1+/- mice). Heterozygote (Notch1+/-) and wild-type (Notch1+/+) transgenic mice were inoculated with Moloney MuLV and the frequency of Notch1 rearrangements was compared between both groups. Notch1 was rearranged at similar frequencies in both groups, indicating that the full-length Notch1 allele is dispensable in tumors harboring an activated Notch1 allele.

Involvement of Notch1 in the development of mouse mammary tumors.

The MMTV/neu transgenic (Tg) mice spontaneously develop mammary tumors stochastically after a long latent period, suggesting that the c-neu/erbB2 oncogene is not sufficient for tumor formation. To identify putative collaborator(s) of the c-neu/erbB2, we used the provirus insertional mutagenesis approach with mammary tumors arising in MMTV/neu Tg mice infected with the mouse mammary tumor virus (MMTV). The Notch1 gene was identified as a novel target for MMTV provirus insertional activation. In Notch1-rearranged tumors, the Notch1 gene was interrupted by the MMTV provirus insertion upstream of the exons coding for the TM domain. These insertions led to overexpression of novel 5' truncated approximately 7 kb RNA coding for 280 kDa mutant protein harboring only the Notch1 ectodomain, N(EC)mut. These may be involved in tumor formation. Another consequence of these insertions was the expression of truncated 3' Notch1 transcripts (3.5 - 4.5 kb) and proteins (86 - 110 kDa) deleted of most of the extracellular sequences (Notch1intra). We found that 3' truncated Notch1intra can transform HC11 mouse mammary epithelial cells in vitro. Deletion analysis revealed that the ankyrin-repeats and the domain 1 (aa 1751 - 1821) are required, while a signal peptide, the two conserved cysteines (C1652 and C1685) and the OPA and PEST sequences are dispensable for transformation. These results indicate that the N-terminally truncated Notch1intra protein behaves as an oncogene in this system.